Painful Diabetic Neuropathy Is Associated with Compromised Microglial IGF-1 Signaling Which Can Be Rescued by Green Tea Polyphenol EGCG in Mice

Background Painful diabetic neuropathy (PDN) is a frequent and troublesome complication of diabetes, with little effective treatment. PDN is characterized by specific spinal microglia-mediated neuroinflammation. Insulin-like growth factor 1 (IGF-1) primarily derives from microglia in the brain and serves a vital role in averting the microglial transition into the proinflammatory M1 phenotype. Given that epigallocatechin-3-gallate (EGCG) is a potent anti-inflammatory agent that can regulate IGF-1 signaling, we speculated that EGCG administration might reduce spinal microglia-related neuroinflammation and combat the development of PDN through IGF-1/IGF1R signaling. Methods Type 1 diabetes mellitus (T1DM) was established by a single intraperitoneal (i.p.) injection of streptozotocin (STZ) in mice. The protein expression level of IGF-1, its receptor IGF1R, interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) was determined by Western blot or immunofluorescence. Results The spinal IGF-1 expression markedly decreased along with the presence of pain-like behaviors, the spinal genesis of neuroinflammation (increased IL-1β, TNF-α, and Iba-1+ microglia), and the intensified M1 microglia polarization (increased iNOS+Iba-1+ microglia) in diabetic mice. IGF-1 could colocalize with neurons, astrocytes, and microglia, but only microglial IGF-1 was repressed in T1DM mice. Furthermore, we found that i.t. administration of mouse recombinant IGF-1 (rIGF-1) as well as i.t. or i.p. treatment with EGCG alleviated the diabetes-induced pain-like behaviors, reduced neuroinflammation (suppressed IL-1β, TNF-α, and Iba-1+ microglia), prevented the M1 microglia polarization (less iNOS+Iba-1+ microglia), and restored the microglial IGF-1 expression. Conclusions Our data highlighted the importance of maintaining spinal IGF-1 signaling in treating microglia-related neuroinflammation in PDN. This study also provides novel insights into the neuroprotective mechanisms of EGCG against neuropathic pain and neuroinflammation through IGF-1 signaling, indicating that this agent may be a promising treatment for PDN in the clinical setting.


Introduction
Painful diabetic neuropathy (PDN) is a frequent and troublesome complication of diabetes, disturbing about one in four diabetic patients [1,2]. The incidence of PDN is even rising over time [3], owing to the increasing diabetic popula-tion [4]. The characteristic clinical symptom of PDN comprises various unpleasant experiences such as mechanical allodynia [5], significantly reducing life quality and raising health care costs. Even opiates and antidepressants are employed for neuropathic pain; their utilization for PDN is limited due to the unbearable side effects and unsatisfactory  2 Oxidative Medicine and Cellular Longevity response rates [6,7]. Thus, investigation of applicable treatments for PDN is urgently needed. Neuroinflammation in the spinal cord has been recognized as a vital process in neuropathic pain [8,9]. One feature of neuroinflammation is the induction of activated microglia and reactive astrocytes that exaggerate the release of cytokines, chemokines, or toxic compounds [10,11]. Specifically, researches on PDN found a comparable extent of neuroinflammation in the spinal cord and ascribe it to the activation of microglia rather than astrocytes [12,13], indicating a specific role of microglia in PDN. The activated microglia include two functional phenotypes: the classically activated (M1) phenotype and the alternative activated (M2) phenotype [14,15]. In response to pathogens or tissue injury, the resting (M0) microglia transits into the M1 phenotype that produces proinflammatory mediators (i.e., tumor necrosis factor-α (TNF-α) and interleukin 1 beta (IL-1β)) or toxic compounds [14,15], whereas M2 microglia are associated with anti-inflammatory properties [14,15]. Therefore, it was reasonable to assume that blocking the transition into M1 microglia or enhancing the M2 phenotype might attenuate PDN by reducing spinal neuroinflammation.
Insulin-like growth factor 1 (IGF-1) is an anabolic growth hormone providing a crucial role in brain growth, maturation, and neuroplasticity [16]. The IGF-1 receptor (IGF1R) expresses abundantly in the developing brain and remains highly active in the adult nervous system [17]. Clinical studies suggest that low IGF-1 levels are associated with diabetes and obesity [18][19][20]. It is also noted that IGF-1/ IGF1R signaling has been implicated in microglia-mediated neuroinflammation. Some studies reported that IGF-1 mainly derives from microglia in comparison with astrocytes and neurons in the central nervous system [21,22]. Further in vitro researches suggested direct anti-inflammatory effects of IGF-1 on microglia [23]. Several lines of evidence indicate that IGF-1 may also regulate the microglial phenotype: the increase of IGF-1 represses the M1 phenotype and promotes the M2 phenotype [22,24]. A recent study also demonstrated that activating IGF1R with recombinant IGF-1 (rIGF-1) reduced neuroinflammation by enhancing M2 microglial polarization after cerebral hemorrhage in mice [20]. These results suggest that IGF-1/IGF1R signaling is a promising target for reducing spinal microglia-related neuroinflammation in PDN.
Green tea is a widely popular beverage, and its customary intake has gradually been reported with health benefits [25,26]. (−)-Epigallocatechin-3-gallate (EGCG), the major bioactive component of green tea, exhibits robust antioxidant and neuroprotective effects against stroke [27] and neurodegenerative diseases [28]. EGCG supplementation can prevent the progression of diabetes [29] and alleviate maternal diabetes-induced neural dysfunction [30] in rodents. Interestingly, studies also demonstrated that EGCG is of strong anti-inflammatory properties by reducing microgliainduced neuroinflammation in the animal models of obesity [31] and stroke [27]. This anti-inflammatory effect of EGCG has been partly attributed to the action of suppressing neurotoxic M1 microglia and enhancing the anti-inflammatory M2 phenotype [20,27]. EGCG is identified as a strong modulator of IGF-1/IGF1R signaling in cancer diseases [32]. However, the detailed effect of EGCG on IGF-1 and its    Oxidative Medicine and Cellular Longevity associated microglia function in PDN remains to be defined. Hence, we speculated that EGCG administration might attenuate spinal microglia-related neuroinflammation and combat the development of PDN through IGF-1/IGF1R signaling.
In the present study, we established the diabetes model in C57BL/6J mice by streptozotocin (STZ) injection and showed that spinal IGF-1/IGF1R signaling and particularly microglial IGF-1 diminished along with pain-like behaviors, spinal neuroinflammation, and augmented M1 microglial polarization. These alterations could be relieved by intrathecal (i.t.) administration of recombinant IGF-1 (rIGF-1) and EGCG. Our data highlighted the importance of maintaining spinal IGF-1 signaling in treating microglia-related neuroinflammation in PDN. This study also provides novel insights into the neuroprotective mechanisms of EGCG against neuropathic pain and neuroinflammation through IGF-1 signaling, indicating that this agent may be a promising treatment for PDN in the clinical setting.

Materials and Methods
2.1. Animals. All experimental protocols and animal handling procedures were approved by Sun Yat-Sen University and conformed to the regulations on animal care accredited by the National Institutes of Health and the institutional animal ethical committee. Male C57BL/6J mice, weighing 25~30 g, were purchased from the Laboratory Animal Center of Guangdong Province (Guangzhou, China) and kept in a standard lab housing with a 12 h light/dark cycle at a temperature of 21 ± 2°C and 60-70% humidity and allowed access to standard diet and water ad libitum.

Induction of Diabetes in Mice.
We induced diabetes by a single i.p. injection of freshly prepared streptozotocin (STZ) (150 mg/kg; Sigma-Aldrich, Germany) in sterile 0.1 M sodium citrate buffer (pH 4.5) [33]. Control mice were only injected i.p. with vehicle solution (sterile 0.1 M sodium citrate buffer, pH 4.5). The successful establishment of T1DM was confirmed by determining the fasting blood glucose level with Accu-Chek test strips (Roche Diagnostics, Indianapolis, IN, U.S.A.) 3 days after the STZ injection. All the STZ-injected mice presented with high levels of blood glucose (>16.7 mmol/L). No mouse died through the study period or required insulin supplementation to offset extreme weight loss, and at the study end, all STZ-injected mice remained hyperglycemic (fasting blood glucose > 16:7 mmol/L).

Drug
Administration. The intrathecal injection method was performed as previously described [34,35]. In brief, mice were covered with a soft towel and held gently but firmly by the hip bones via the thumb and index finger of the nondominant hand of the operator. A 5 μL microsyringe (Hamilton Company, Nevada, USA; 30 G needle) was inserted at the midline of the iliac crest between the lumbar 5th and 6th vertebrae. This region is considered the level of the cauda equina. A reflexive flick of the tail indicates the  The cell number of IGF1 + NeuN + , IGF1 + GFAP + , or IGF1 + Iba-1 + in the SDH. * * P < 0:01. Data are expressed as the mean ± SD. n = 4/group. (f) IGF-1 + ratio in Iba-1 + cells in the SDH on D14. * * * * P < 0:0001. Data are expressed as the mean ± SD. n = 4/group. (g) The quantitative analysis of Iba-1 + microglia in the SDH on D14. * * P < 0:01. Data are expressed as the mean ± SD. n = 4/group. (h) The area of GFAP + immunoreactivity in the SDH on D14. Data are expressed as the mean ± SD. n = 4 /group. GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium-binding adaptor protein-1; IGF-1: insulin-like growth factor 1; IGF1R: insulin-like growth factor 1 receptor; ns: not significant; control: the mice treated with vehicle (0.1 M sodium citrate buffer); SDH: the spinal dorsal horn; T1DM: the mice treated with STZ (diabetic mice). 5 Oxidative Medicine and Cellular Longevity successful puncture of the dura. Because this reaction and muscle tone are essential reflections, intrathecal injections were performed in conscious mice. After achieving a 90% to 95% success rate in training sessions, we started these experiments. According to our recent study [36], mouse rIGF-1 (Novoprotein, Shanghai, China) was diluted in saline and administered intrathecally (i.t.) 1 μg/d from D3 (3 days after STZ injection) to D5 after STZ injection, while the sham group was i.t. injected with equal volume of saline. EGCG (Sigma-Aldrich, St. Louis, MO, USA) was diluted in saline and administered i.t. 2 μg/d [27] or i.p. 20 mg/kg/d [37] from D3 to D5 after STZ injection.     7 Oxidative Medicine and Cellular Longevity and D21 (n = 9/group). Western blot and immunofluorescence were conducted on lumbar spinal cord tissues at D14 (n = 5/group).

Behavioral Tests.
On the experiment day, the mice were placed individually in transparent test compartments with a wire mesh bottom and habituated for one hour. Paw mechanical withdrawal thresholds (PMWTs) in response to mechanical stimuli were evaluated using the electronic von Frey unit (Bioseb, Montpellier, France) with a flexible metal filament applying increasing force (from 0 to 10 g) against the plantar surface of the hind paw of the mouse [38]. The nocifensive paw withdrawal response automatically turned off the stimulus, and the mechanical pressure that evoked the response was recorded. Measurements were repeated 5 times, and the final value was obtained by averaging the 5 measurements.
Thermal hypersensitivity was assessed by measuring paw thermal withdrawal latency (PTWL) to thermal stimuli using the PL-200 Plantar Analgesia Tester (Chengdu Tech-nology & Market Co., Ltd., Sichuan, China) as described previously [39,40]. The mice were placed on a glass plate and were allowed to habituate to the apparatus for 30 min. The radiant heat lamp source was adjusted to position right beneath the hind paw's plantar surface, vertically projecting a light spot with a diameter of 5 mm. The PTWL was calculated by averaging three individual trials with 5 min intervals to avert unexpected thermal sensitization. A cutoff time of 12 s was set to avoid tissue injury.
2.6. Western Blot. The mice were sacrificed by anesthetic overdose (2.5% Avertin, 1600 mg/kg, i.p.). The lumbar enlargement (L4-L5) of the spinal cord was rapidly removed and homogenized in ice-cold RIPA buffer (Beyotime, Shanghai, China). The lysates were kept for 30 min and then centrifuged at 14,000 g for 10 min at 4°C. Supernatants were collected, and the protein concentration was determined by the BCA protein assay kit (Boster, Wuhan, China). Equal amounts of protein samples (50 μg) from each group were separated using 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (Millipore, Bedford, MA, USA) membranes. Then, the membranes were blocked with 3% nonfat milk in TBST for 1 h at room temperature and then incubated with the primary antibodies overnight at 4°C. On the next day, the membranes were rinsed three times with TBST and incubated with secondary antibody (antimouse or rabbit IgG, 1 : 5000; Boster) for 2 h at room temperature. After washing three times with TBST, the protein bands were detected with enhanced chemiluminescent reagents (Beyotime) and analyzed in the Fluochem HD2 Imaging System (Alpha Innotech, USA). The densitometry of the blots was analyzed with ImageJ software (version 1.43) and normalized to β-actin. The experimenters were blinded to the group assignment.

Spinal IGF-1/IGF1R Signaling Diminished along with the Development of Pain-Like Behaviors in STZ-Injected Diabetic
Mice. We induced the diabetic model by injecting mice with a single dose of STZ (150 mg/kg, i.p.). To assess the time course of pain-like behaviors, we conducted the electronic von Frey test before and after the STZ injection. Our data showed that, compared with the vehicle (0.1 M sodium citrate buffer)-treated mice, the paw mechanical withdrawal thresholds (PMWTs) of T1DM (STZ-injected) mice began to decline on D3 (3 days after STZ injection) and reached the bottom on D14 and lasted to D21 (Figure 1(a); T1DM vs. control, F ð1, 14Þ = 364:1, P < 0:0001, n = 8), signifying the development of mechanical allodynia in the T1DM mice. We also determined the thermal nociception behaviors after the STZ injection. We found that compared with the vehicletreated mice, the paw thermal withdrawal latency (PTWL) of T1DM mice dropped after STZ injection (Figure 1(b); T1DM vs. control, F ð1, 14Þ = 155:7, P < 0:0001, n = 8), indicating the occurrence of thermal hypersensitivity in T1DM mice. CGRP is a neuropeptide widely distributed in nociceptive circuits and is closely involved in pain transmission [41]. The immunostaining of the SDH suggested that the CGRP immunoreactivity of T1DM mice was more intense than that of control mice (Figure 1(c)).
In addition, we analyzed the Iba-1 + microglia number and GFAP + immunoreactivity area in the SDH of control and T1DM mice. We found that compared with control mice, the Iba-1 + microglia number in the SDH of T1DM mice was significantly higher (Figure 2(g); T1DM vs. control, 78:13 ± 16:77 vs. 28:99 ± 10:41/mm 2 , P = 0:0025; n = 4 ), whereas the difference in the GFAP + immunoreactivity area was not significant (Figure 2(h), P = 0:6457; n = 4). Based on these results and the previous evidence that microglia are indispensable for spinal neuroinflammation in PDN [43], we postulated that the inhibition of IGF-1/ IGF1R signaling might play a crucial role in spinal microglia activation of diabetic mice.

3.3.
Activated M1 Phenotype Microglia and Neuroinflammation Surged in the SDH of STZ-Injected Diabetic Mice. The switch between M1 and M2 microglial phenotypes regulates inflammation in the central nervous system [44,45]. Thus, we investigated the alteration of M1 (iNOS + ) [46] and M2 (Arg-1 + ) [47] microglia phenotypes in the spinal cord. Our immunostaining results showed that the number and ratio of iNOS + Iba-1 + microglia increased in the SDH of T1DM mice compared with control mice (Figure 3(a); P = 0:002 for the number of iNOS + Iba-1 + cells, P = 0:0024 for iNOS + ratio in Iba-1 + microglia, n = 4). In contrast, the Arg-1 + Iba-1 + microglia were not detectable by immunostaining in the SDH of either control or T1DM mice. Thus, we sought to detect the protein expression of iNOS and Arg-1 by Western blot in the spinal cord. The data revealed that the iNOS protein expression was upregulated in T1DM mice on D14 (Figure 3(b); P = 0:0007, n = 5) while Arg-1 was not altered (Figure 3(b); P = 0:1708, n = 5), suggesting a significant increase of M1 microglia in the spinal cord of diabetic mice. M1 microglia are characterized by releasing proinflammatory cytokines such as IL-1β and TNF-α. The further detection of IL-1β and TNF-α corroborated the activation of M1 microglia by showing that IL-1β and TNF-α protein expressions are elevated in the T1DM mice (Figure 3(c); P = 0:0135 for IL-1β, P = 0:0006 for TNF-α, n = 5).

Discussion
Our study identified the dysfunction of IGF-1 signaling, especially microglial IGF-1, as an essential mechanism underlying the spinal neuroinflammation in PDN. We also revealed the potency of ECGG in maintaining microglia IGF-1 signaling and reducing neuroinflammation in PDN. Specifically, we found that the spinal IGF-1 expression markedly declined parallel to the development of pain-like behaviors, neuroinflammation (increased IL-1β, TNF-α, and Iba-1 + microglia), and the increased M1 microglia polarization (increased iNOS + Iba-1 + microglia) in diabetic mice. In the spinal cord, IGF-1 could colocalize with neurons, astrocytes, and microglia, but only microglial IGF-1 was suppressed in T1DM mice. Furthermore, we found that i.t. administration of rIGF-1 as well as i.t. or i.p. treatment with EGCG alleviated the diabetes-induced pain-like behaviors, reduced neuroinflammation, averted the M1 microglia polarization, and recovered the microglial IGF-1 expression.
Neuroinflammation served a significant role in peripheral nerve injury-induced neuropathic pain [49,50]. Increasing evidence reveals that nonneuronal glia cells contribute largely to the neuroinflammation in the key region regulating pain (i.e., the SDH) [49,50]. Our recent study found a significant rise in reactive astrocytes and microglia in the animal model of chronic constriction injury of the sciatic nerve [35]. However, studies have demonstrated a particular role of microglia activation in the SDH of diabetic animals, signifying a characteristic role of microglia in PDN. This was verified in the present study that the GFAP + immunoreactivity was not altered while Iba-1 + microglia were extensively increased in the SDH of diabetic mice.
IGF-1 is a protein that has various functions in the CNS, including neuronal survival and synapse growth [16]. Generally, IGF-1 is considered a neuroprotective molecule which is at least partly ascribed to its anti-inflammatory property [22]. Consistent with this, a decrease in IGF-1 signaling has been related to neurodegeneration, depressive disorders, and other brain diseases [22]. Also, intrathecal administration of IGF-1 could induce a central antinociceptive effect and reduce neuroinflammation in the spinal cord of normal rats [51]. This had led us to speculate a potential role of IGF-1 for treating PDN and the associated spinal neuroinflammation.
As expected, we observed that IGF-1 signaling diminished in the spinal cord of T1DM mice, as indicated by the decrease in the protein expression of IGF-1 and p-IGF1R. The downregulation of spinal IGF-1 signaling might be associated with the inflammatory reaction, as the compromised spinal IGF-1 signaling was accompanied by the induction of neuroinflammation (increased IL-1β, TNF-α, and Iba-1 + microglia) and the intensified M1 microglia polarization (increased iNOS + Iba-1 + microglia). We further corroborated the relationship by displaying that the intrathecal administration of rIGF-1 alleviated pain-like behaviors and neuroinflammation in the spinal cord of diabetic mice. These results robustly suggested a potential beneficial effect of maintaining spinal IGF-1 signaling by suppressing neuroinflammation.
Previous studies have reported that IGF-1 can express in neurons, astrocytes, and microglia in the human brain, and its main source is microglia [22]. Indeed, we found that IGF-1 immunostaining colocalized with neurons, astrocytes, and microglia, but the largest proportion was astrocytes rather than microglia in the lumbar spinal cord of diabetic mice. These results are consistent with our recent animal studies in nerveinjury-induced neuropathic pain [35] and chemotherapy-induced peripheral neuropathy [36]. However, we noted that merely microglial IGF-1 was significantly suppressed in diabetic mice while the alterations in neurons and astrocytes were absent, implying a critical role of impaired microglial IGF-1 signaling underlying the pathogenesis of PDN.
IGF-1 has long been recognized in regulating microglial phenotypes [22,24]. Some studies indicate that IGF-1 + microglia represent the anti-inflammatory M2 phenotype [22,24], while others argue that IGF-1 could be present in resting (M0) microglia [52]. In this regard, we detected microglial M1 and M2 phenotypes by immunofluorescence in the spinal cord and showed that the main proportion of activated Iba-1 + microglia were the M1 phenotype (iNOS + ) while the M2 microglia (Arg-1 + ) were rare in the SDH of either control or diabetic mice. Additional Western blot data verified the result by demonstrating that spinal iNOS protein expression was markedly increased in diabetic mice but without obvious alteration in Arg-1. Combined with the observed decrease of IGF-1 + microglia in diabetic mice, we thought that IGF-1 + microglia were more likely to denote the resting microglia rather than the M2 phenotype. This result is in agreement with a study on spinal cord injury and experimental autoimmune encephalomyelitis, which demonstrated that spinal Arg-1 + cells are not resident but mainly derived from macrophages migrating from responsive immune organs [53].
Although we had revealed the benefit of rIGF-1 supplementation for PDN, caution is still needed for expanding its applications in neuropathic pain. One of our current studies showed that IGF-1 signaling overactivation contributed to the development of neuropathic pain caused by peripheral nerve injury [35], implying that overdose rIGF-1 might even aggravate PDN. EGCG, a potent component of green tea, has been reported with robust